Molecular Plant Breeding 2024, Vol.15, No.5, 209-219 http://genbreedpublisher.com/index.php/mpb 212 Additionally, the genes selected for stacking should have complementary modes of action to avoid cross-resistance. For example, combining genes that produce different types of toxins can be more effective than using multiple genes that produce the same toxin (Gressel et al., 2017; Salim et al., 2020). The goal is to create a robust defense system that pests find difficult to overcome, thereby extending the effectiveness of the resistance traits. In conclusion, multi-gene stacking is a powerful strategy in citrus pest resistance breeding, leveraging advanced genetic technologies and a deep understanding of pest resistance mechanisms to develop durable and effective pest-resistant crops. By carefully selecting and combining multiple genes, researchers can create crops that are better equipped to withstand pest pressures and reduce the reliance on chemical pesticides. 4 Genetic Basis of Pest Resistance in Citrus 4.1 Key genes involved in citrus pest resistance The genetic basis of pest resistance in citrus involves several key genes that have been identified and utilized in breeding programs. One notable example is the resistance gene CsLOB1, which has been successfully used to induce resistance to citrus bacterial canker in sweet orange and grapefruit through genome editing techniques (Salonia et al., 2020). This gene plays a crucial role in enhancing the plant's defense mechanisms against bacterial pathogens. Additionally, other genes such as those involved in the synthesis of secondary metabolites and structural proteins contribute to the overall pest resistance in citrus. 4.2 Interaction of these genes with pest biology The interaction between pest resistance genes and pest biology is complex and multifaceted. For instance, the CsLOB1 gene in citrus not only provides resistance to bacterial canker but also interacts with the pest's ability to infect and proliferate within the host plant (Salonia et al., 2020). The effectiveness of these genes often depends on their expression levels, the timing of expression, and the specific tissues in which they are active. Moreover, the presence of multiple resistance genes can create a more robust defense system, making it harder for pests to overcome the plant's defenses. This multi-gene approach can delay the evolution of pest resistance, as pests would need to simultaneously adapt to multiple defense mechanisms (Gressel et al., 2017). 4.3 Strategies for identifying and validating resistance genes Identifying and validating resistance genes in citrus involves several strategies. One common approach is the use of new plant breeding techniques (NPBTs) such as genome editing and cisgenesis, which allow for precise modifications of the plant genome to introduce or enhance resistance traits (Salonia et al., 2020). These techniques require a thorough understanding of the genes controlling specific traits and their interactions with pest biology. Another strategy is the use of transgene stacking systems, which involve the assembly of multiple resistance genes into a single construct and their introduction into the plant genome via transformation methods. This approach has been successfully used in other crops, such as rice, to develop multi-resistance varieties (Li et al., 2020). The validation of these genes typically involves field trials and laboratory assays to assess their effectiveness against target pests and their impact on plant health and yield. In summary, the genetic basis of pest resistance in citrus is underpinned by key resistance genes such as CsLOB1, which interact with pest biology to provide effective defense mechanisms. Strategies for identifying and validating these genes include the use of NPBTs and transgene stacking systems, which have shown promise in enhancing pest resistance in citrus and other crops (Gressel et al., 2017; Li et al., 2020; Salonia et al., 2020). 5 Application of Multi-Gene Stacking in Citrus Breeding 5.1 Design and construction of gene stacks for citrus pest resistance The design and construction of gene stacks for citrus pest resistance involve the strategic combination of multiple genes that confer resistance to various pests and diseases. This approach leverages advanced genetic engineering techniques to introduce and express multiple resistance genes within a single citrus plant. For instance, the use of CRISPR/Cas9 technology has been instrumental in modifying susceptibility genes such as CsLOB1 to confer resistance to citrus canker (Jia et al., 2017). Additionally, the integration of resistance genes from related species
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